J Assist Reprod Genet (2015) 32:225–231 DOI 10.1007/s10815-014-0385-y
ASSISTED REPRODUCTION TECHNOLOGIES
Uterine flushing with supernatant embryo culture medium in vitrified warmed blastocyst transfer cycles: a randomized controlled trial Mohan S. Kamath & Mariona Mascarenhas & Kanchanadevi B & Nidhi N. Vasani & Asmita Joshi & Muthukumar K & Korula George
Received: 1 September 2014 / Accepted: 3 November 2014 / Published online: 27 November 2014 # Springer Science+Business Media New York 2014
Abstract Purpose Does transfer of supernatant embryo culture fluid (stimulation of endometrial embryo transfer - SEET) prior to vitrified warmed blastocyst transfer result in better clinical pregnancy and live birth rates than direct vitrified warmed blastocyst transfer? Methods This randomized controlled trial compared SEET group and direct transfer group (control) in 60 women undergoing vitrified warmed blastocyst transfers. The duration of the study was 3 years. The patients were undergoing vitrified warmed blastocyst transfer at university level infertility centre. Sixty women were randomized to SEET (n=30) or control (n=30). Results Data was available for analysis from all the 30 women in the SEET group and 30 women in the control group. There were no drop outs in the trial. The implantation rate was significantly lower in the SEET group compared to the control group (27 vs. 44 %, P=0.018). The clinical pregnancy rates were similar in both the groups (47 vs. 53 %) but the live birth rate was also significantly lower in SEET group (23 vs. 50 %, P=0.03). Limitations The sample size based on clinical pregnancy rates was small and hence not adequately powered to detect
Capsule In women undergoing vitrified warmed blastocyst transfer, the clinical pregnancy rate following prior transfer of supernatant embryo culture fluid (SEET) did not improve compared to direct transfer. TRIAL REGISTRATION NUMBER: CTRI/2013/01/003280. M. S. Kamath : M. Mascarenhas : K. B : N. N. Vasani : A. Joshi : M. K Reproductive Medicine Unit, Christian Medical College Hospital, Vellore, Tamil Nadu 632004, India K. George (*) Reproductive Medicine Unit, Bangalore Baptist Hospital, Hebbal Bangalore 560024, India e-mail: [email protected]
differences in live birth rates. Lack of blinding leading to possible bias cannot be ruled out. Conclusion There was no evidence of an improvement in clinical pregnancy rate following SEET in vitrified warmed blastocyst transfer compared to direct transfer. Keywords SEET . Supernatant embryo culture medium . Blastocyst transfer
Introduction Successful implantation of embryos in the uterine cavity is the end result of a synchronized sequence of complex physiological events [1]. This coordinated development of the embryo and uterine endometrium, ultimately leading to an optimal environment for implantation, is possible due to the existence of a communication link between the maternal tissue and embryo, commonly known as “cross-talk”. There is evidence to suggest that this “cross talk” is mainly carried out through secretion of various factors by the developing embryo [2]. They include interleukins [3], immunosuppressive factors [4] and growth factors which have been isolated from the culture fluid surrounding the embryo during in-vitro culture. These factors produced by the embryo induce endometrial expression [5] of integrins, leukemia inhibitory factor and other factors which possibly facilitate implantation. Currently in Assisted Reproductive Technology (ART) practice, blastocyst stage transfer compared to cleavage stage transfer is associated with higher live birth rates [6]. Worldwide following blastocyst transfer, the implantation rate has been around 4–55 % and the rate has remained stagnant in the past decade [6]. A proposed reason for this lower implantation rate is the absence of cross-talk till the embryo is transferred [7].
226
With an intention to introduce cross talk prior to a delayed embryo transfer, which occurs in day 5 blastocyst transfers, Goto et al. tried a novel method called stimulation of endometrial embryo transfer (SEET). In this trial supernatant embryo culture fluid from the fresh embryo cycle, was transferred prior to a frozen blastocyst transfer in poor prognosis patients resulting in a significantly higher pregnancy rates [7]. However a similar study conducted in patients undergoing cleavage stage embryo transfer, did not show any benefits [8]. Due to conflicting nature of the results, we decided to study the effectiveness of the SEET protocol following transfer of vitrified-warmed blastocysts.
Materials and methods We decided to evaluate whether transferring the supernatant embryo culture prior to warmed blastocyst transfer resulted in better clinical outcomes when compared to direct vitrified warmed blastocyst transfer. To test this hypothesis, we planned a randomized controlled trial and invited all consecutive women who were due for a fresh blastocyst transfer and had supernumerary embryos available for cryopreservation. For eligible women who agreed to participate in the trial, we cryopreserved the supernatant culture medium along with the supernumerary blastocysts. The study duration was three years (Sep 2011 to June 2014). Women entering the trial were randomly distributed, using a computer generated randomization sequence (blocks of 6), into two groups – the study (SEET) and control group. The randomization sequence was generated by a statistician from the institutional biostatistics department. Allocation concealment was achieved by using consecutively numbered opaque sealed envelope. Once the women were planned for vitrified thawed transfer, the envelope was opened and group allotment was done by the investigators. In women allotted to the SEET group thawed supernatant embryo culture fluid was injected into the uterine cavity two days prior to the planned blastocyst transfer. In the control group, direct vitrified warmed blastocyst transfer was planned. The study was done at university level infertility centre in India and was approved by the institutional review board. The trial was registered with the clinical trial registry of India (CTRI/2013/01/003280). ART protocol: Prior to the study cycle, all patients underwent a fresh ART cycle using either the agonist or antagonist protocol. Controlled ovarian hyperstimulation (COH) was done using recombinant gonadotrophins and oocyte retrieval was planned 35 h after human chorionic gonadotrophin (hCG) trigger administration. Fertilization was achieved either by In Vitro Fertilization (IVF) or Intra Cytoplasmic Sperm Injection (ICSI). Fertilized oocytes were transferred into cleavage medium (SAGE cleavage medium, Trumbull, Connecticut, USA), incubated and observed for cleavage on day 3.
J Assist Reprod Genet (2015) 32:225–231
On day 3, if four or more grade 1 embryos (8 cells stage with no or minimal fragmentation) were obtained, they were transferred into blastocyst medium (SAGE blastocyst medium, Trumbull, Connecticut, USA) and cultured till day 5. Fresh blastocyst transfer was performed on day 5, and supernumerary good quality blastocysts were chosen for vitrification. Embryos were graded according to Gardner’s grading system and embryos with a score of 3AA or more were considered good quality [9]. These blastocysts were cryopreserved on day 5 and day 6. Vitrification and warming protocol Supernumerary blastocysts were vitrified using the solid surface method. A pre-cooled metal block (Cryologic, Victoria, Australia), fibre plugs (Cryologic, Victoria, Australia) and in house prepared equilibrium and vitrification solution were used. The equilibrium solution was composed of ethylene glycol and dimethyl sulphoxide in HEPES HTF medium (Cryobase, Cook IVF, Queensland, Australia) [10]. Warming of vitrified blastocysts was done using filter sterilized trehalose solution and blastocyst medium (SAGE Blastocyst medium, Trumbull, Connecticut, USA). Survival was assessed by the percentage of viable trophoectodermal and inner cell mass cells together with the degree of reexpansion of the blastocoel cavity. Assisted hatching using laser was performed prior to transfer. After transfer of embryos in fresh cycle, for women who had supernumerary blastocysts and who were willing to participate in the trial, the supernatant culture medium was transferred into a sterile Eppendorf container and frozen at −80 °C. Transfer of blastocysts /supernatant culture fluid and assessment of outcome Endometrial preparation for vitrified thawed blastocyst transfer was done by starting step-wise increment of estradiol valerate (Progynova, Schering AG, Germany; 2 mg once daily from Day 1–5, 2 mg daily twice from Day6-9 and daily three times from Day 10–15). On day 15 a transvaginal ultrasound was done to check for endometrial thickness and to rule out follicular activity. Micronized progesterone (Orgagest vaginal pessaries, Schering AG, Germany; 400 mgs daily twice) was started once endometrial thickness of greater than 7 mm was documented. In the SEET group, 10–15 μl of thawed supernatant embryo culture medium was injected into the uterine cavity transcervically, using an embryo transfer catheter placed just beyond the internal os, two days prior to the vitrified thawed blastocyst transfer. For both the groups, the vitrified thawed blastocyst transfer was done on day 6 after initiation of progesterone.
J Assist Reprod Genet (2015) 32:225–231
After pre transfer counseling, between one to three surviving blastocysts were transferred. A serum beta hCG was done on the 12th day following embryo transfer. Women with a positive pregnancy test were advised to continue luteal support and a transvaginal ultrasound was carried out ten days later to confirm clinical pregnancy and viability. Once confirmed, antenatal care was provided and women were followed up till delivery. Hormonal supplementation was stopped at 12 weeks of gestation. Clinical pregnancy rate was defined as clinical pregnancy (ultrasound visualization of gestational sac) per embryo transfer. Live birth rate was defined as number of deliveries with at least one live born baby per embryo transfer. All enrolled women underwent the study cycle only once. The primary outcome was the clinical pregnancy rate while secondary outcomes were implantation rate, multiple pregnancy rates, miscarriage and live birth rates. It was an open label trial. The vitrified blastocyst embryo pregnancy rate per transfer in our centre was 40 %. (Goto et al. in their study demonstrated a doubling of the clinical pregnancy rate following SEET transfer (48 to 87 %). Hypothesizing a doubling of the clinical pregnancy rate (80 %) following SEET transfer, a sample size of 30 women in each arm (80 % power and alpha 0.05 for a two sided test) was calculated. Data obtained was analyzed using SPSS version 15 (Chicago, IL). Independent t test was used for analyzing continuous data. χ2 test and Fishers exact test was used for categorical data. The differences were considered to be significant if P
ASSISTED REPRODUCTION TECHNOLOGIES
Uterine flushing with supernatant embryo culture medium in vitrified warmed blastocyst transfer cycles: a randomized controlled trial Mohan S. Kamath & Mariona Mascarenhas & Kanchanadevi B & Nidhi N. Vasani & Asmita Joshi & Muthukumar K & Korula George
Received: 1 September 2014 / Accepted: 3 November 2014 / Published online: 27 November 2014 # Springer Science+Business Media New York 2014
Abstract Purpose Does transfer of supernatant embryo culture fluid (stimulation of endometrial embryo transfer - SEET) prior to vitrified warmed blastocyst transfer result in better clinical pregnancy and live birth rates than direct vitrified warmed blastocyst transfer? Methods This randomized controlled trial compared SEET group and direct transfer group (control) in 60 women undergoing vitrified warmed blastocyst transfers. The duration of the study was 3 years. The patients were undergoing vitrified warmed blastocyst transfer at university level infertility centre. Sixty women were randomized to SEET (n=30) or control (n=30). Results Data was available for analysis from all the 30 women in the SEET group and 30 women in the control group. There were no drop outs in the trial. The implantation rate was significantly lower in the SEET group compared to the control group (27 vs. 44 %, P=0.018). The clinical pregnancy rates were similar in both the groups (47 vs. 53 %) but the live birth rate was also significantly lower in SEET group (23 vs. 50 %, P=0.03). Limitations The sample size based on clinical pregnancy rates was small and hence not adequately powered to detect
Capsule In women undergoing vitrified warmed blastocyst transfer, the clinical pregnancy rate following prior transfer of supernatant embryo culture fluid (SEET) did not improve compared to direct transfer. TRIAL REGISTRATION NUMBER: CTRI/2013/01/003280. M. S. Kamath : M. Mascarenhas : K. B : N. N. Vasani : A. Joshi : M. K Reproductive Medicine Unit, Christian Medical College Hospital, Vellore, Tamil Nadu 632004, India K. George (*) Reproductive Medicine Unit, Bangalore Baptist Hospital, Hebbal Bangalore 560024, India e-mail: [email protected]
differences in live birth rates. Lack of blinding leading to possible bias cannot be ruled out. Conclusion There was no evidence of an improvement in clinical pregnancy rate following SEET in vitrified warmed blastocyst transfer compared to direct transfer. Keywords SEET . Supernatant embryo culture medium . Blastocyst transfer
Introduction Successful implantation of embryos in the uterine cavity is the end result of a synchronized sequence of complex physiological events [1]. This coordinated development of the embryo and uterine endometrium, ultimately leading to an optimal environment for implantation, is possible due to the existence of a communication link between the maternal tissue and embryo, commonly known as “cross-talk”. There is evidence to suggest that this “cross talk” is mainly carried out through secretion of various factors by the developing embryo [2]. They include interleukins [3], immunosuppressive factors [4] and growth factors which have been isolated from the culture fluid surrounding the embryo during in-vitro culture. These factors produced by the embryo induce endometrial expression [5] of integrins, leukemia inhibitory factor and other factors which possibly facilitate implantation. Currently in Assisted Reproductive Technology (ART) practice, blastocyst stage transfer compared to cleavage stage transfer is associated with higher live birth rates [6]. Worldwide following blastocyst transfer, the implantation rate has been around 4–55 % and the rate has remained stagnant in the past decade [6]. A proposed reason for this lower implantation rate is the absence of cross-talk till the embryo is transferred [7].
226
With an intention to introduce cross talk prior to a delayed embryo transfer, which occurs in day 5 blastocyst transfers, Goto et al. tried a novel method called stimulation of endometrial embryo transfer (SEET). In this trial supernatant embryo culture fluid from the fresh embryo cycle, was transferred prior to a frozen blastocyst transfer in poor prognosis patients resulting in a significantly higher pregnancy rates [7]. However a similar study conducted in patients undergoing cleavage stage embryo transfer, did not show any benefits [8]. Due to conflicting nature of the results, we decided to study the effectiveness of the SEET protocol following transfer of vitrified-warmed blastocysts.
Materials and methods We decided to evaluate whether transferring the supernatant embryo culture prior to warmed blastocyst transfer resulted in better clinical outcomes when compared to direct vitrified warmed blastocyst transfer. To test this hypothesis, we planned a randomized controlled trial and invited all consecutive women who were due for a fresh blastocyst transfer and had supernumerary embryos available for cryopreservation. For eligible women who agreed to participate in the trial, we cryopreserved the supernatant culture medium along with the supernumerary blastocysts. The study duration was three years (Sep 2011 to June 2014). Women entering the trial were randomly distributed, using a computer generated randomization sequence (blocks of 6), into two groups – the study (SEET) and control group. The randomization sequence was generated by a statistician from the institutional biostatistics department. Allocation concealment was achieved by using consecutively numbered opaque sealed envelope. Once the women were planned for vitrified thawed transfer, the envelope was opened and group allotment was done by the investigators. In women allotted to the SEET group thawed supernatant embryo culture fluid was injected into the uterine cavity two days prior to the planned blastocyst transfer. In the control group, direct vitrified warmed blastocyst transfer was planned. The study was done at university level infertility centre in India and was approved by the institutional review board. The trial was registered with the clinical trial registry of India (CTRI/2013/01/003280). ART protocol: Prior to the study cycle, all patients underwent a fresh ART cycle using either the agonist or antagonist protocol. Controlled ovarian hyperstimulation (COH) was done using recombinant gonadotrophins and oocyte retrieval was planned 35 h after human chorionic gonadotrophin (hCG) trigger administration. Fertilization was achieved either by In Vitro Fertilization (IVF) or Intra Cytoplasmic Sperm Injection (ICSI). Fertilized oocytes were transferred into cleavage medium (SAGE cleavage medium, Trumbull, Connecticut, USA), incubated and observed for cleavage on day 3.
J Assist Reprod Genet (2015) 32:225–231
On day 3, if four or more grade 1 embryos (8 cells stage with no or minimal fragmentation) were obtained, they were transferred into blastocyst medium (SAGE blastocyst medium, Trumbull, Connecticut, USA) and cultured till day 5. Fresh blastocyst transfer was performed on day 5, and supernumerary good quality blastocysts were chosen for vitrification. Embryos were graded according to Gardner’s grading system and embryos with a score of 3AA or more were considered good quality [9]. These blastocysts were cryopreserved on day 5 and day 6. Vitrification and warming protocol Supernumerary blastocysts were vitrified using the solid surface method. A pre-cooled metal block (Cryologic, Victoria, Australia), fibre plugs (Cryologic, Victoria, Australia) and in house prepared equilibrium and vitrification solution were used. The equilibrium solution was composed of ethylene glycol and dimethyl sulphoxide in HEPES HTF medium (Cryobase, Cook IVF, Queensland, Australia) [10]. Warming of vitrified blastocysts was done using filter sterilized trehalose solution and blastocyst medium (SAGE Blastocyst medium, Trumbull, Connecticut, USA). Survival was assessed by the percentage of viable trophoectodermal and inner cell mass cells together with the degree of reexpansion of the blastocoel cavity. Assisted hatching using laser was performed prior to transfer. After transfer of embryos in fresh cycle, for women who had supernumerary blastocysts and who were willing to participate in the trial, the supernatant culture medium was transferred into a sterile Eppendorf container and frozen at −80 °C. Transfer of blastocysts /supernatant culture fluid and assessment of outcome Endometrial preparation for vitrified thawed blastocyst transfer was done by starting step-wise increment of estradiol valerate (Progynova, Schering AG, Germany; 2 mg once daily from Day 1–5, 2 mg daily twice from Day6-9 and daily three times from Day 10–15). On day 15 a transvaginal ultrasound was done to check for endometrial thickness and to rule out follicular activity. Micronized progesterone (Orgagest vaginal pessaries, Schering AG, Germany; 400 mgs daily twice) was started once endometrial thickness of greater than 7 mm was documented. In the SEET group, 10–15 μl of thawed supernatant embryo culture medium was injected into the uterine cavity transcervically, using an embryo transfer catheter placed just beyond the internal os, two days prior to the vitrified thawed blastocyst transfer. For both the groups, the vitrified thawed blastocyst transfer was done on day 6 after initiation of progesterone.
J Assist Reprod Genet (2015) 32:225–231
After pre transfer counseling, between one to three surviving blastocysts were transferred. A serum beta hCG was done on the 12th day following embryo transfer. Women with a positive pregnancy test were advised to continue luteal support and a transvaginal ultrasound was carried out ten days later to confirm clinical pregnancy and viability. Once confirmed, antenatal care was provided and women were followed up till delivery. Hormonal supplementation was stopped at 12 weeks of gestation. Clinical pregnancy rate was defined as clinical pregnancy (ultrasound visualization of gestational sac) per embryo transfer. Live birth rate was defined as number of deliveries with at least one live born baby per embryo transfer. All enrolled women underwent the study cycle only once. The primary outcome was the clinical pregnancy rate while secondary outcomes were implantation rate, multiple pregnancy rates, miscarriage and live birth rates. It was an open label trial. The vitrified blastocyst embryo pregnancy rate per transfer in our centre was 40 %. (Goto et al. in their study demonstrated a doubling of the clinical pregnancy rate following SEET transfer (48 to 87 %). Hypothesizing a doubling of the clinical pregnancy rate (80 %) following SEET transfer, a sample size of 30 women in each arm (80 % power and alpha 0.05 for a two sided test) was calculated. Data obtained was analyzed using SPSS version 15 (Chicago, IL). Independent t test was used for analyzing continuous data. χ2 test and Fishers exact test was used for categorical data. The differences were considered to be significant if P
